Hemostatic materials

ABSTRACT

A safe and effective hemostatic material is provided. A hemostatic material comprising as an effective ingredient thrombin and fibrinogen characterized in that a bioabsorbable synthetic nonwoven fabric is used as a supporting material. The use of the hemostatic material of the present invention allows for quick and thorough hemostasis for both projectile bleeding and exudative bleeding.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a hemostatic material comprising as aneffective ingredient thrombin and fibrinogen characterized in that abioabsorbable synthetic nonwoven fabric is used as a supportingmaterial.

BACKGROUND OF THE INVENTION

Hemostatic management is very important on the clinical scene. Inparticular, in case of surgical operation, hemostatic manipulation isone of the most momentous tasks requiring much labor and time. As anexemplary, it is reported that a little longer two hours out of aboutfive hours of operation may be used for hemostatic manipulation in aradical operation of tetralogy of Fallot. Thus, shortening of time forhemostatic manipulation will much shorten time for operation.

Moreover, for hemostasis are important not only shortening of time forhemostasis but also suppression of a loss of blood to a minimum extentbefore hemostasis is achieved as well as thorough hemostasis. Bleeding,if in excess, will be lethal or cause severe complications subsequently.If hemostasis is not thoroughly attained, bleeding will repeatedly occurduring or after operation. Recurrence of bleeding during operation evenat a low degree may prevent a visual field for surgical operation torender easy manipulation more difficult. Recurrence of bleeding afteroperation may increase drainage from a drainage tube and liability toinfection to thereby make postoperative management difficult. In worstcases, surgical operation will become necessary for hemostasis. In thefield of cardiovascular surgery, there is a report that 3% of patientsare forced to undergo surgical operation for hemostasis. Accordingly,shortening of time for operation and suppression of loss of blood aswell as thorough hemostasis have been earnestly desired so thatphysicians' burden and stress of patients may be lowered.

A method for hemostasis commonly used includes (1) a method forhemostasis using a mechanical means and (2) a method for hemostasis byaccelerating topical coagulation. Usually, a topical hemostatic or afilling agent is used for topical hemostasis in case that a method usinga mechanical means such as ligature or suture may not effectively beapplied. When a hemostatic is used, collagen fiber, dry thrombin powder,oxidized cellulose, gelatins, fibrin adhesives, etc. may be delivered oradhered to the bleeding site, which is then compressed for five to tenminutes. With the currently used method for hemostasis, there areproblems as described above that substantial time for hemostasis must betaken from time for surgical operation, that ligature may be slippedoff, that recurrence of bleeding may occur due to non-thoroughhemostasis, and the like.

DISCLOSURE OF THE INVENTION TECHNICAL PROBLEM TO BE SOLVED BY THEINVENTION

Among the conventional hemostatic measures is hemostasis with a fibrinadhesive. Specifically, a fibrin adhesive is used for adhesion, sealingand hemostasis of tissues by overlaying fibrin and thrombin solutions onwounded regions or by applying a mixed solution of fibrin and thrombinwith a spraying device. However, using a fibrin adhesive alone forhemostasis of arterial bleeding, which is vigorous and may lead to muchloss of blood, efficacious hemostasis is scarcely possible since saidmixed ingredients in liquid may tend to be flowed away by the bloodflow.

There are attempts to fix the ingredients of a fibrin adhesive onto avariety of substrates to thereby produce a sheet-like preparation. Forsuch substrates, bioabsorbable/biodegradable materials have been usedincluding natural components such as gelatin or collagen, or synthetichigh molecular weight materials such as polyethylene glycol orpolyglycolic acid. As an exemplary, a sheet preparation has been putinto practice wherein horse-derived collagen holds fibrin and thrombin(e.g. Japanese patent publication No. 34830/1986). However, thesubstrate collagen of this sheet preparation is rather thick andsomewhat rigid to render the sheet preparation poorly stick to woundedregions where hemostasis is desired, thereby making efficacioushemostasis sometimes difficult. Besides, said sheet preparation is suchthat the substrate is made of equine collagen and thrombin is derivedfrom bovine, i.e. material derived from non-human animal species isused, and hence there is a possibility of induction of an antibodyagainst heterologous proteins or onset of zoonotic infections such asprion disease, being far from ideal one. On the other hand, the use of afibrin adhesive for hemostasis will advantageously induce a fewerreaction to foreign substance but has the problems as previouslydescribed.

In order to solve these problems, one approach is to develop a fibrinadhesive that may permit hemostasis in a short time, suppress a loss ofblood to a minimum extent and allow for thorough hemostasis. Such afibrin adhesive will be required to consist of the same coagulationfactor as in human free from infectious agents, to be in the form of asheet so that hemostatic effect may fully be exerted, and to use a sheetmade of a material strictly selected and devised to be safe to theliving body.

MEANS FOR SOLVING THE PROBLEMS

In view of the above-mentioned various problems, the present inventorshave carried out intensive investigation and as a consequence found thata hemostatic material comprising as an effective ingredient thrombin andfibrinogen characterized in that a bioabsorbable synthetic nonwovenfabric, which is a bioabsorbable synthetic material processed in theform of a nonwoven fabric, is used as a supporting material may exertquite excellent hemostatic effects, to thereby complete the presentinvention.

MORE EFFICACIOUS EFFECTS THAN PRIOR ART

The hemostatic material according to the present invention has excellentproperties as listed below and hence is an ideal topical hemostaticmaterial: (1) It may be applied to bleeding under various conditions,including projectile bleeding and exudative bleeding, suppress a loss ofblood to a minimum extent, and provide a thorough hemostasis; (2) It ishighly safe; (3) It is absorbed with a lapse of time; (4) It shows anexcellent elasticity and flexibility; (5) It allows for hemostasis at abroad area; (6) It induces a slight or no inflammation reaction.

In accordance with the present invention, it is now possible to providefor a hemostatic material comprising a bioabsorbable synthetic nonwovenfabric which enables safe, prompt and thorough hemostasis in variousclinical fields, typically in a surgical operation in various fields ofthe operation.

BEST MODE FOR CARRYING OUT THE INVENTION

The bioabsorbable synthetic nonwoven fabric for use in the presentinvention may be any nonwoven fabric made of a bioabsorbable syntheticfiber. A bioabsorbable synthetic fiber as used herein refers to asynthetic fiber that is unlikely to induce inflammation in the livingbody as a foreign substance and may be absorbed and/or degraded withinthe living body with a lapse of time. The nonwoven fabric has preferablyappropriate flexibility and elasticity to ensure that it may surely bestuck to any affected area. For example, a synthetic fiber that may formsuch a nonwoven fabric includes polyglycolic acid, polylactic acid, or acopolymer of glycolic acid with lactic acid, etc., which may be usedafter processing into a nonwoven fabric. Among these, a bioabsorbablesynthetic nonwoven fabric which is prepared from polyglycolic acid byprocessing into a nonwoven fabric is the most preferable material forthe purpose of the present invention.

The nonwoven fabric may be in any shape but preferably in the form of asheet in view of versatility to various applications.

In addition to the effective ingredients, a pharmaceutically acceptablestabilizer and additive may also be added. Examples of such stabilizerand additive include, for instance, Factor XIII preferably derived fromhuman blood or obtained by the genetic recombination technique, calciumchloride, a protease inhibitor (e.g. aprotinin), albumin, aminoaceticacid, polyethylene glycol, arginine, sodium hyaluronate, glycerol,mannitol, and the like.

Thrombin, fibrinogen and Factor XIII may preferably be derived fromhuman blood or obtained by the genetic recombination technique.

The hemostatic material of the present invention may be in any dosageform so far as thrombin and fibrinogen as an effective ingredient areultimately contained in a bioabsorbable synthetic nonwoven fabric.

In view of easy handling under operative settings, however, abioabsorbable synthetic nonwoven fabric previously holding thrombin,which maintains flexibility, is one of preferable embodiments from theviewpoint of its easy handling as well as hemostatic efficacy.

In case that a bioabsorbable synthetic nonwoven fabric previously holdsboth thrombin and fibrinogen, the nonwoven fabric should hold each ofthrombin and fibrinogen under such condition that the components areseparated from each other or each of the components in the form ofpowder are suspended in an organic solvent and each suspension issprayed to the nonwoven fabric, so that both thrombin and fibrinogen maynot react to each other before use to generate stabilized fibrin.

The hemostatic material of the present invention may be formulated as akit comprising either: (i) a bioabsorbable synthetic nonwoven fabricholding thrombin plus fibrinogen; or (ii) a bioabsorbable syntheticnonwoven fabric, thrombin, and fibrinogen; in which a stabilizer and anadditive as described above may optionally be added to both (i) and(ii).

For use in case of (i), a bioabsorbable synthetic nonwoven fabricholding thrombin is immersed into a solution containing fibrinogen, orsaid solution is sprayed to the bioabsorbable synthetic nonwoven fabricholding thrombin. Said bioabsorbable synthetic nonwoven fabric holdingthrombin may be prepared by (1) dissolving thrombin in a saline or abuffer and optionally adding to the resulting thrombin solution calciumchloride as an additive, and (2) immersing a bioabsorbable syntheticnonwoven fabric into said thrombin solution, followed by freezing at−80° C. for 2 hours and lyophilization.

For use in case of (ii), a solution containing thrombin and a solutioncontaining fibrinogen are prepared as in the process for preparing acommercially available fibrin adhesive (e.g. Bolheal manufactured byJuridical Foundation The Chemo-Sero-Therapeutic Research Institute) anda bioabsorbable synthetic nonwoven fabric is then immersed into thesolutions of thrombin and fibrinogen successively, or each of thesolutions of thrombin and fibrinogen is applied simultaneously viaspray.

In either case of. (i) or (ii), Factor XIII or a protease inhibitor maybe added to a solution containing fibrinogen.

The hemostatic material obtained in accordance with the presentinvention, due to its high adhesiveness, appropriate strength,flexibility and elasticity, may be stuck to bleeding regions in anyshape, allowing for prompt hemostasis for various bleeding conditionssuch as projectile bleeding and exudative bleeding.

Polyglycolic acid bioabsorbable nonwoven fabric as used for thesubstrate in the hemostatic material of the present invention is highlysafe since it is absorbed within the living body and degraded into waterand carbon dioxide.

As such, the hemostatic material according to the present invention mayeasily and quickly be applied to topical bleeding and allow forefficient hemostasis through both pressure and a blood coagulationreaction. Besides, since every material used therein is safe to theliving body, it may be used in clinical settings without care.

The present invention is explained in more detail by means of thefollowing Examples but should not be construed to be limited thereto.

EXAMPLE 1 Preparation of Sheet Holding Thrombin

A sheet holding thrombin in accordance with the present invention wasprepared by the process as described below.

To a solution containing 5% human serum albumin are added 40 mM calciumchloride and subsequently thrombin derived from human blood at a finalconcentration of 500 U/mL. The solution is poured into a vessel at adepth of 1 mm where a bioabsorbable synthetic nonwoven fabric made ofpolyglycolic acid (Neoveil, Gunze Limited, thickness 0.15 mm) is laid onthe bottom. The sheet, after being frozen at −80° C. for 2 hours andlyophilized, is used as a sample of a sheet holding thrombin (thrombinheld at 50 U/cm²).

EXAMPLE 2 Test for Hemostasis in Projectile Bleeding

Hemostatic effect to projectile bleeding was investigated for acombination of the sheet holding thrombin as prepared in Example 1 and afibrinogen solution.

Assessment used is indicated below.

(1) Test rabbit was subject to abdominal section under anesthesia withNembutal (20 to 35 mg/kg).

(2) Heparin was intravenously administered at 300 U/kg.

(3) The abdominal aorta was stuck with 21 G needle to generateprojectile bleeding.

(4) Attempt was made to cease bleeding for each of Groups with varioushemostatic means as described below. Hemostasis was conducted whileblood spouting.

Group 1: Sheet Holding Thrombin+Fibrinogen Solution

A fibrinogen solution (Bolheal manufactured by Juridical Foundation TheChemo-Sero-Therapeutic Research Institute, “A solution”; about 0.7 mL)was sprayed to the sheet holding thrombin (2×2 cm) prepared in Example1, which was immediately placed onto the bleeding region and lightlypressed for 1 minute.

Group 2: Polyglycolic Acid Nonwoven Fabric+Fibrin Adhesive

To the polyglycolic acid bioabsorbable synthetic nonwoven fabric(Neoveil, Gunze Limited, 2×2 cm) as used in Example 1 as a substrate wassprayed a fibrin adhesive (Bolheal, Juridical Foundation TheChemo-Sero-Therapeutic Research Institute, a solution containingfibrinogen (“A solution”) and a solution containing thrombin (“Bsolution”); each about 0.7 mL), which was immediately placed onto thebleeding region and lightly pressed for 1 minute.

Group 3: Collagen Sheet Preparation

A collagen sheet preparation in which components of a fibrin adhesiveare fixed (TachoComb, Torii Pharmaceutical Co., Ltd.; fibrinogen andthrombin components are fixed by lyophilization on one side of a spongesheet made of equine collagen as a supporting material: 2×2 cm) wasplaced onto the bleeding region and lightly pressed for 1 minute.

(5) After hemostatic treatment, whether hemostasis was attained or notwas determined. When bleeding still occurred, the same hemostatictreatment was repeated for at most three times.

(6) After completion of hemostasis, if there is recurrence of bleedingwas observed for five minutes.

As a result, as shown in Table 1, Group 1 of a combination of a sheetholding thrombin and a fibrinogen solution could thoroughly ceaseprojectile bleeding with a single hemostatic treatment in this model.Group 2 of a polyglycolic acid nonwoven fabric to which a fibrinadhesive was sprayed was also proved to exhibit an excellent hemostaticeffect though at a slightly lower level than that of Group 1 as having acase where bleeding could not be ceased with a single hemostatictreatment. On the contrary, with a collagen sheet of Group 3, bleedingcould be ceased with a single hemostatic treatment in only few cases.Recurrence of bleeding after completion of hemostasis was observed inneither of Groups 1 to 3. TABLE 1 Nos. of bleeding Nos. of treatmentsGroup regions 1st 2nd 3rd 1 7 7 0 0 2 7 6 1 0 3 7 2 3 2

EXAMPLE 3 Test for Hemostasis in Exudative Bleeding

Hemostatic effect to exudative bleeding (oozing) was investigated for acombination of the sheet holding thrombin prepared in Example 1 and afibrinogen solution.

Assessment used is indicated below.

(1) Test rabbit was subject to abdominal section under anesthesia withNembutal (20 to 35 mg/kg).

(2) Heparin was intravenously administered at 300 U/kg.

(3) The right lobe, the inner left lobe or the outer left lobe of theliver was wounded in circle to thickness of 4 mm with a leather punch of1.5 cm diameter and the wounded region was excised with a surgicalknife.

(4) Bleeding from the wound was absorbed with gauze for 10 seconds andweighed (0.68 g). No difference in an amount of bleeding was observedamong Groups.

(5) Hemostatic treatment was performed in the same Groups 1 to 3 as inExample 2.

(6) Bleeding for 5 minutes, including the time required for thehemostatic treatment, was absorbed with gauze and weighed. When bleedingfrom the wound surface was observed after 5 minutes, the hemostatictreatment and the weighing of bleeding were repeated.

(7) The hemostatic treatment was repeated for at most three times andassessment was made with a frequency of the hemostatic treatment neededfor hemostasis and a total weight of bleeding from the initiation of thehemostatic treatment up till hemostasis.

As a result, as shown in Table 2, Group 1 of a combination of a sheetholding thrombin and a fibrinogen solution could completely ceaseexudative bleeding with a single hemostatic treatment in this model andbleeding after hemostatic treatment (an amount of bleeding up tillhemostasis) could be suppressed to an extremely low level. With apolyglycolic acid nonwoven fabric to which a fibrin adhesive was sprayedof Group 2, exudative bleeding could be ceased with a single hemostatictreatment although a total bleeding after hemostatic treatment wasobserved at somewhat higher level than the combination of a sheetholding thrombin and a fibrinogen solution. On the contrary, with acollagen sheet of Group 3, bleeding could not be ceased with a singlehemostatic treatment and there were even cases where bleeding could notbe ceased with three hemostatic treatments. Moreover, a high level of atotal bleeding after hemostatic treatment was observed. TABLE 2 Nos. ofTotal bleeding after Nos. of hemostatic hemostatic treatments bleedingtreatments (g/5 min. × Nos. of Group regions 1st 2nd 3rd treatments) 1 77 0 0 0.05 ± 0.033 2 5 5 0 0 0.11 ± 0.055 3 5 0 3  2* 6.41 ± 5.736*Even after the third hemostatic treatment, hemostasis was not possible.

The bioabsorbable synthetic nonwoven fabric coated with fibrinogen andthrombin according to the present invention could provide thoroughhemostasis with hemostatic treatment for as short as 1 minute as shownin Examples 2 and 3.

1. A hemostatic material comprising as an effective ingredient thrombinand fibrinogen held on a supporting material consisting of abioabsorbable synthetic nonwoven fabric.
 2. The hemostatic materialaccording to claim 1, wherein said bioabsorbable synthetic nonwovenfabric is made of a material selected from the group consisting ofpolyglycolic acid, polylactic acid and a copolymer of glycolic acid andlactic acid.
 3. The hemostatic material according to claim 1, whereinsaid bioabsorbable synthetic nonwoven fabric is a nonwoven fabric madeof a material of polyglycolic acid.
 4. The hemostatic material accordingto claim 1, wherein the bioabsorbable synthetic nonwoven fabricpreviously holds at least thrombin among thrombin and fibrinogen.
 5. Thehemostatic material according to claim 1, wherein said hemostaticmaterial comprises at least one additive selected from Factor XIII, aprotease inhibitor, or calcium chloride.
 6. The hemostatic materialaccording to claim 1, wherein thrombin, fibrinogen and Factor XIII areeither derived from human blood or produced by a genetic recombinationtechnique. 7-13. (canceled)
 14. A method of preparing a bioabsorbablesynthetic nonwoven fabric holding thrombin as an effective ingredient,comprising the steps of immersing a bioabsorbable synthetic nonwovenfabric into a solution containing thrombin and lyophilizing the obtainednonwoven fabric.
 15. The method according to claim 14, wherein saidbioabsorbable synthetic nonwoven fabric is made of a material selectedfrom the group consisting of polyglycolic acid, polylactic acid and acopolymer of glycolic acid and lactic acid.
 16. The method according toclaim 14, wherein said bioabsorbable synthetic nonwoven fabric is anonwoven fabric made of a material of polyglycolic acid.
 17. The methodaccording to any claim 14, wherein said hemostatic material comprises atleast one additive selected from Factor XIII, a protease inhibitor, orcalcium chloride.
 18. The method according to claim 17, wherein saidcalcium chloride is fixed to the bioabsorbable synthetic nonwoven fabrictogether with thrombin.
 19. The method according to claim 17, whereinsaid Factor XIII is added to fibrinogen.
 20. The method according toclaim 14, wherein said thrombin, fibrinogen and Factor XIII are eitherderived from human blood or produced by a genetic recombinationtechnique.
 21. A hemostatic kit comprising a bioabsorbable syntheticnonwoven fabric holding thrombin as an effective ingredient, and acontainer comprising fibrinogen as an effective ingredient.
 22. Thehemostatic kit according to claim 21, wherein said bioabsorbablesynthetic nonwoven fabric is made of a material selected from the groupconsisting of polyglycolic acid, polylactic acid and a copolymer ofglycolic acid and lactic acid.
 23. The hemostatic kit according to claim21, wherein said bioabsorbable synthetic nonwoven fabric is a nonwovenfabric made of a material of polyglycolic acid.
 24. The hemostatic kitaccording to claim 21, wherein said hemostatic kit comprises at leastone additive selected from Factor XIII, a protease inhibitor, or calciumchloride.
 25. The hemostatic kit according to claim 24, wherein saidcalcium chloride is added to the bioabsorbable synthetic nonwoven fabricas an additive for thrombin.
 26. The hemostatic kit according to claim24, wherein said Factor XIII is included in a container comprisingfibrinogen.
 27. The hemostatic kit according to claim 21, wherein saidthrombin, fibrinogen and Factor XIII are either derived from human bloodor produced by a genetic recombination technique.
 28. The hemostatic kitaccording to claim 21, wherein said bioabsorbable synthetic nonwovenfabric holding thrombin is prepared by the steps of immersing abioabsorbable synthetic nonwoven fabric into a solution containingthrombin and of lyophilizing the obtained nonwoven fabric.
 29. Ahemostatic kit comprising a bioabsorbable synthetic nonwoven fabric as asubstrate, a container comprising thrombin as an effective ingredientand a container comprising fibrinogen as an effective ingredient. 30.The hemostatic kit according to claim 29, wherein said bioabsorbablesynthetic nonwoven fabric is made of a material selected from the groupconsisting of polyglycolic acid, polylactic acid and a copolymer ofglycolic acid and lactic acid.
 31. The hemostatic kit according to claim29, wherein said bioabsorbable synthetic nonwoven fabric is a nonwovenfabric made of a material of polyglycolic acid.
 32. The hemostatic kitaccording to claim 29, wherein said hemostatic kit comprises at leastone additive selected from Factor XIII, a protease inhibitor, or calciumchloride.
 33. The hemostatic kit according to claim 32, wherein saidFactor XIII is included in a container comprising fibrinogen.
 34. Thehemostatic kit according to claim 29, wherein said thrombin, fibrinogenand Factor XIII are either derived from human blood or produced by agenetic recombination technique.